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First published online 19 January 2005
doi: 10.1242/dev.01601

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Lunatic fringe null female mice are infertile due to defects in meiotic maturation

Katherine L. Hahn^1,4, Joshua Johnson^2,*, Brian J. Beres^2,4, Sheena Howard^3,4 and Jeanne Wilson-Rawls^1,2,4,

¹ Molecular and Cellular Graduate Program, Arizona State University, Tempe, AZ 85284-4501, USA
² Biology Graduate Program, Arizona State University, Tempe, AZ 85284-4501, USA
³ Minority Access to Research Careers (MARC) Program at ASU, Arizona State University, Tempe, AZ 85284-4501, USA
⁴ School of Life Sciences, Box 4501, Arizona State University, Tempe, AZ 85284-4501, USA

View larger version (98K): [in a new window]   Fig. 1. Lunatic fringe is expressed in the granulosa cells and theca. Whole-mount thick section ISH of wild-type 42-day-old ovary was carried out using antisense digoxigenin-labeled RNA gene-specific probes. (A) Lfng-specific probes including 3' untranslated sequences and Lfng transcripts were detected in follicles from type 3 to preovulatory in granulosa cells, but not in oocytes or interstitial cells. (B) Lfng exon only probe. Transcripts were detected in the theca and in the vasculature (red arrow indicates positive blood vessel), a band of Lfng transcripts surrounding small follicles was observed (inset). (C) Rfng was expressed transiently in the granulosa cells of antral follicles (arrow). (D) Mfng transcripts were only detected in the vasculature (red arrow). (E) RT-PCR from total ovary RNA demonstrates the presence of Mfng and Rfng transcripts. E9.5 represents total E9.5 embryo control; L+/+ represents total ovary RNA from a 42-day-old wild-type animal; mock represents no RT control. (F) RT-PCR of Lfng and Gdf9 transcripts from total RNA from GV and MII stage oocytes. E9.5, total embryo control; MW, 100 bp ladder. Scale bars: 100 µm.

View larger version (108K): [in a new window]   Fig. 2. Histological and morphological examination of Lfng–/– ovaries. (A) Ovaries from 4-week-old Lfng+/– and Lfng–/– mice; null ovaries are smaller and developing follicles are obvious in both. (B) The reproductive tracts of 7-week-old Lfng+/– and Lfng–/– mice; all structures were present and no gross abnormalities were noted. (C,D) Histological sections of neonatal ovaries stained with Hematoxylin and Eosin. Primordial follicles are evident; however, the Lfng null ovary (C) is smaller than the Lfng+/– ovary (D) and the morphology is not as well organized. (E-J) Histological sections of ovaries from 42-day-old Lfng–/– mice. Many abnormal follicles were noted. Some large follicles had smaller follicles within their boundaries (E, black arrow). There were follicles that shared theca or had incomplete theca (F, black arrowheads). There were polyovular follicles containing two or three oocytes (F, red arrows; G,H). There were also many large lutealinized follicles with trapped oocytes (I,J). Scale bars: 100 µm.

View larger version (40K): [in a new window]   Fig. 3. Lfng–/– females ovulate in response to exogenous hormones, but oocytes are not at meiotic metaphase II. (A) After hormone administration, OCC were collected from the oviduct. Oocytes were stained with anti--tubulin-FITC and Hoechst 33258, and visualized using confocal microscopy at 800 x final magnification. Genotypes are as indicated. In Lfng+/+ and Lfng+/– oocytes, note the presence of the polar bodies (white arrowhead) and chromosomes aligned on a metaphase spindle. Many Lfng–/– oocytes contained one or more bodies with scattered chromatin (white arrowheads). Some Lfng–/– oocytes were at anaphase/telophase I; note the lack of a polar body. (B) Representative kinase assays analyzed on 15% SDS-PAGE and visualized using a phosphorimager. Lanes are as marked. MBP, myelin basic protein. (C) Lfng+/– and Lfng–/– mice were induced to ovulate and OCC were collected from the oviducts. OCC demonstrated normal cumulus expansion in Lfng null mutant females, under light microscopy at 70 x magnification. (D) Graphs show relative kinase activity of MPF and CSF, the kinase assays were quantified and expressed as fold activity over control (n=9/genotype) ±s.d.

View larger version (40K): [in a new window]   Fig. 4. sqRT-PCR examination of gene expression in Lfng–/– ovaries. sqRT-PCR with gene-specific primers was performed to detect transcripts in total ovary RNA from 42-day-old mice of all three genotypes (Lfng+/+, Lfng+/– and Lfng–/–). Three samples per genotype were normalized against the ribosomal gene L7 and representative data are presented. N2, Notch2; N3, Notch3; J1, Jagged1; J2, Jagged2; EP2, prostaglandin type 2 receptor (Ptger2 – Mouse Genome Informatics); Fshr, follicle stimulating hormone receptor.

View larger version (78K): [in a new window]   Fig. 5. Expression of Notch family genes in the Lfng–/– ovary by ISH. Gene-specific digoxigenin labeled probes were used, as indicated; representative data from a minimum of three replicates of Lfng+/– and Lfng–/– are presented. Notch3 demonstrated no change in follicle stage or cell-type-specific expression in null ovaries; black arrows indicate granulosa cell staining and the red arrowhead indicates expression in an early CL. Jagged1 was limited to oocytes of small growing follicles in the Lfng–/– ovary (arrowhead). Expression of Notch downstream target genes Hes5, Hes1, Hesr1 and Hesr2 was not detected in the Lfng–/– follicles (only representative family members Hes5 and Hesr2 are presented). Note positive follicles indicated by arrows in +/– and compare with similarly sized follicles indicated by arrowheads in –/–.

View larger version (76K): [in a new window]   Fig. 6. Expression of other follicle-specific genes. Connexin43 (Cx43) was detected by IHC using an alkaline phosphatase-conjugated secondary antibody and DAB substrate, as indicated by the red stain in granulosa cells and oocytes. There was no alteration in Cx43 expression. Kit ligand is expressed normally in Lfng–/– granulosa cells. Kit ligand transcripts were detected by ISH, note the purple stain in granulosa cells, dark field microscopy. Kit receptor (c-Kit) was also unaffected in Lfng null follicles. Kit receptor was detected by IHC, arrowhead in +/– indicates a group of primary follicles with positive oocytes. Controls included no primary antibody, note the lack of red stain.

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